Developing techniques for quantitatively evaluating chemical substances generated or consumed in cells significantly contributes not only to development of fundamental biochemistry but also to the medical and life science fields, such as cytoscreening used for cancer screening tests and the like, quality evaluation of transplantation cells used in regenerative medicine, immune cell therapies and the like, and for use as substitutes for experimentation on animals for drug efficacy assessments and toxicity assessments.
However, bioactivities of cells vary with environments surrounding the cells, such as temperature, pH, medium compositions, adjacent cells, and extracellular matrixes and also vary over time depending on external stimuli such as gene introduction, drug exposure, application of stress and the like and cell events such as cell division and cell death.
Therefore, in order to evaluate true properties of cells that actually act in a biological body, it is important to place sample cells alive (while maintaining cell bioactivities) in an environment that is as close to an intravital environment as possible and to measure a chemical substance generated or consumed in the cells in real-time with respect to external stimuli and cell events.
One widely-used method for placing sample cells in an environment that is close to an intravital environment is to select as a sample a cell aggregate (spheroid) which is an aggregate of multiple cells and extracellular matrix (ECM) components, rather than a single cell.
This is because many of various bioactivities of cells undergo interaction with adjacent cells and ECM that contact the cells and therefore a cell aggregate which is an aggregate of them may replicate an intravital environment more faithfully.
Such cell aggregates may include spheroids of pancreatic islet cells obtained from pancreas, fertilized eggs, liver cells and nerve cells obtained through cell culture and embryoid bodies of embryonic stem (ES) cells.
While such cell aggregates have diameters that differ depending on the types of component cells, regions in a biological body from which the cell aggregates were obtained, culture conditions and the like, cell aggregates having diameters of about 100 to 600 μm are often used in evaluation of cell activities. This is because the number of component cells in a small cell aggregate with a diameter of 100 μm or smaller is too small for bioactivities specific to cell aggregates to appear whereas in a large cell aggregate having a diameter of 600 μm or greater, oxygen does not diffuse to cells in the central part of the cell aggregate and cell necrosis is likely to occur.
As an approach to measuring a chemical substance generated or consumed in cells in real-time, an electrochemical approach is used. The electrochemical approach requires electrodes (working electrodes) that are placed in the same solution together with a sample and used for detecting various electrochemical signals from the sample. There are various detection methods with variations in potential control or current control of the working electrodes. In measurement relating to metabolic activities of cells or the like, potentiostatic electrolysis (constant-potential electrolysis) exemplified by chronoamperometry and cyclic voltammetry have been used because of its high comparison performance and simplicity of analysis. In the potentiostatic electrolysis, the potential of a working electrode is controlled as a function of time and a current value that appears in the working electrode is detected during the control.
In electrochemical measurement of a chemical substance generated or consumed in a common cell aggregate, a reaction system that causes a chemical substance having a redox activity to be generated inside the cell aggregate or at the surface of the cell aggregate in association with substance metabolism of the cell aggregate is incorporated and is oxidized or reduced on a working electrode to generate a current.
While various systems can be designed that depend on a metabolic system of interest as reaction systems that cause a chemical substance having a redox activity to be generated in association with substance metabolism of cells, systems that use an enzyme reaction are popularly used among others for the purpose of detecting a trace amount of metabolic substance with high sensitivity.
For example, in an embryoid body which is a cell aggregate made from mouse ES cells, the amount of alkaline phosphatase (ALP) which is an enzyme existing at the surface of cells increases or decreases depending on the differentiation state of the embryoid body.
In order to evaluate the differentiation state of an embryoid body, electrochemical evaluation of the amount of generated ALP is often performed (Non-patent literature 1). In the evaluation system, an embryoid body is placed in a solution in which p-aminophenyl phosphate (PAPP), which is a substrate substance, is dissolved and a dephosphorylation is facilitated by ALP enzyme activity, thereby generating p-aminophenol (PAP) having redox activity.
When the PAPP concentration in the solution is sufficiently high, the amounts of PAP, which is a redox-active chemical substance, can be accumulated with time by the enzymic activity of PAP even though the amount of ALP generated from cells is ultralow. Consequently, the amount of existing ALP can be detected with high sensitivity.